Summary The objective is to understand the organizing principles of the skin for future use in regenerative medicine. Progress has been made toward generating skin progenitor cells. However, before significant progress in clinical applications can be made, we must know how the skin exhibits region-specific characteristics with unique functions and architectures. While human skins show regional specificity (scalp, face, palm, etc), mice do not. Chicken skin does show dramatic regional specificity and is accessible to experimentation, making it an ideal model for this type of study. Tissue recombination studies showed dermis controls the phenotypes, and epidermal progenitors respond by making appropriate appendage phenotypes. In this proposal, we will focus on how regional specific keratinocyte differentiation is controlled by epigenetic processes. Our recent work showed that chicken ?-keratin clusters on Chr25 are organized in five sub-clusters, each enriched in a globally different skin region (feather, scale, claw, ?macro-regional specificity?); whereas the ?-keratin clusters on Chr27 are differentially expressed in different within-feather regions (rachis vs barb) or feather generations (downy vs adult contour feathers) made from stem cells in the same follicle (?micro- regional specificity?). Thus, we hypothesize there is a hierarchical correspondence between the skin regional topographic map and genomic organization of b-keratin clusters. Preliminary data of Chr25 keratins show typical enhancers in front of keratin sub-clusters, while Chr27 keratins surprisingly show 38 peaks of CTCF/KLF4 binding motifs, strongly suggesting intra-cluster chromatin looping and complex combination potential. In Aim 1, we will use histone ChIP-Seq to identify how typical enhancer(s) differentially regulate the b-keratin cluster within the EDC in different skin regions. We will also analyze how intra-cluster higher-order chromatin looping in the Chr27 keratin cluster may lead to differential keratin expression in within-feather differences. Feather and scales at different competent stages, tissue recombinant explants with reprogrammed epithelial fate, and experimental conditions in which scales are converted toward feathers, and adult contour feathers converted toward downy feathers will be used to monitor the epigenetic changes. In Aim 2, we will analyze how the genome organizers CTCF, KLF4, and SATB1/2 bind DNA and configure chromatin conformation at the keratin loci in the above conditions. Appendage phenotypes caused by suppression of these genome organizers will be analyzed.